Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore

The Journal of Cell Biology
Rakefet Ben-YishayYaron Shav-Tal

Abstract

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

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Citations

Sep 5, 2020·Essays in Biochemistry·Jianshu Wang, Hong Cheng
Jan 9, 2020·The Biochemical Journal·Asaf Ashkenazy-TitelmanRalph H Kehlenbach
Jul 29, 2020·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Sarah E Hasenson, Yaron Shav-Tal
Nov 23, 2019·The Journal of Cell Biology·Carina Patrizia DerrerElisa Dultz
Sep 13, 2020·Nature Communications·Vasilisa AksenovaMary Dasso
Apr 4, 2021·Viruses·Daniela Dünn-KittenplonRonit Sarid
Jul 21, 2021·Biochemical Society Transactions·Alexandr A MakarovMark C Field

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Methods Mentioned

BETA
transfection
confocal microscopy
FRET
superresolution microscopy
PCR
transfections
FCS
immunoprecipitation

Software Mentioned

FV1000
LAS X
STED
Excel
dis
MATLAB
FRAP
fit
Imaris
Prism

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